SciELO - Scientific Electronic Library Online

 
vol.32 issue2Genetic parameters for faecal egg count, packed-cell volume and body-weight in Santa Inês lambsKaryology of the Atlantic forest rodent Juliomys (Cricetidae): a new karyotype from southern Brazil author indexsubject indexarticles search
Home Pagealphabetic serial listing  

Services on Demand

Journal

Article

Indicators

Related links

Share


Genetics and Molecular Biology

Print version ISSN 1415-4757On-line version ISSN 1678-4685

Genet. Mol. Biol. vol.32 no.2 São Paulo  2009

http://dx.doi.org/10.1590/S1415-47572009000200015 

ANIMAL GENETICS
RESEARCH ARTICLE

 

Characterization analysis and polymorphism detection of the porcine Myd88 gene

 

 

Xinyun LiI, II; Huazhen LiuI; Shulin YangII; Zhonglin TangII; Yuehui MaII; Mingxing ChuII; Kui LiII

IThe Key Laboratory of Animal Genetics, Breeding and Reproduction of Ministry of Education of China, Huazhong Agricultural University, Wuhan, P.R. China
IIThe Key Laboratory for Farm Animal Genetic Resources and Utilization of Ministry of Agriculture of China, Institute of Animal Science, Beijing, P.R. China

Send correspondence to

 

 


ABSTRACT

The myeloid differentiation primary response protein 88 (Myd88) is an essential adaptor protein, which mediates in all Toll-like receptor (TLR) members signal transduction, except for TLR3. In this study, the 4464 bp genomic sequence of porcine Myd88 was first isolated, whereupon tissue distribution, chromosome mapping and single nucleotide polymorphism (SNP) were analyzed. Our results revealed that porcine Myd88 gene, which was located at chromosome 13 linked with marker S0288 (distance = 40 cR; LOD = 8.66), was widely expressed in all the examined tissues. There were 16 potential SNPs in the isolated genome fragment. SNP 797T/C in the first intron was studied, with no significant association being found between the genotype and immune traits in pigs (p > 0.05). The porcine Myd88 protein contained both the death domain (DD) and the Toll/IL-1 receptor domain (TIR). Leu residues, essential for its structure, were the most abundant encountered in the DD. The TIR contained two conserved motifs which may play important roles in the Myd88 function.

Key words: Myd88, TLR, polymorphism, pig, chromosome mapping.


 

 

Introduction

Myd88 is an essential cytoplasmic adaptor protein, critical for Toll-like receptor signal transduction. TLRs play very important roles in host immune reaction defense against invading of microbial pathogens (Lemaitre et al., 1996; Medzhitov and Janeway, 1997). So far, 11 TLR (TLR1-11) members have been characterized (Hardiman et al., 1996; Medzhitov and Janeway, 1997; Palladino et al., 2007). Myd88 is associated with all TLR signaling pathways except for that of TLR3 (Li et al., 2005).

The Myd88 protein contains a Toll/IL-1 receptor domain (TIR) in its C-terminus and a death domain (DD) in its N-terminus (Uematsu and Akira, 2006). All TLRs contain TIR in their cytoplasmic domain. On stimulation, TLRs recruit Myd88 through the TIR-TIR interaction. Myd88 recruits downstream molecular IL-1 receptor kinase (IRAK) to TLRs through the DD-DD interaction. Four IRAK members (IRAKs) have been identified so far, these being IRAK1, IRAK2, IRAK-M and IRAK4. IRAK1 and IRAK4 are activated via phosphorylation in response to stimuli. The downstream molecule, tumor necrosis factor receptor-associated factor 6 (TRAF6), is then activated by IRAKs. Subsequently, TRAF6 activates growth factor-β- activated protein kinase 1 (TAK1) in a ubiquitin-dependent manner. Finally, TAK1 activates the IKK complex, which leads to activation of the NF-κB transcription factor. This TLR signaling pathway is called the Myd88-dependent pathway (Takeda and Akira, 2004; Yamamoto and Akira, 2004). It is essential for the expression of inflammatory cytokines, including TNFα, IL-6, IL-12, IL-1β, as well as co-stimulatory molecules (Adachi et al., 1998; Takeda and Akira, 2004). Inflammatory reactions activated by these inflammatory cytokines are responsible for the removal of invading pathogens, these including bacteria, viruses and protozoans (Adachi et al., 1998; Takeda and Akira, 2004; Yamamoto and Akira, 2004). Previous studies indicated that the expression level or mutations of the Myd88 gene are related to important phenomena such as endotoxin tolerance (Li et al., 2000; Medvedev et al., 2002). Myd88 deficient mice present defects in T cell proliferation, thereby lacking in response to IL-1 and IL-18 (Adachi et al., 1998). They also displayed low resistance to protozoan infection (Scanga et al., 2002). Thus, Myd88 plays very important roles in inflammatory reactions and host defense against infections. Consequently, porcine Myd88 may be an important candidate gene for disease-resistance breeding.

In this study, we first isolated the genomic DNA sequence of the porcine Myd88 gene. We then analyzed tissue distribution, chromosome mapping, polymorphisms and structure characterization. We also studied one SNP in the first intron of porcine Myd88 by the polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP) method. Association analysis with pig immune traits indicated that there was no significant association in our experimental group. Our results provide useful information for further studies on the porcine Myd88 gene.

 

Materials and Methods

Isolation of the porcine Myd88 gene

For genomic DNA isolation, DNA fragments (TI Nos: 768175941; 773990928; 775670732; 847811271; 853168567; 854250486; 857233111; 861225716; 1420071428; 1420478383; 1420500667) of the porcine Myd88 gene were retrieved from GenBank through sequence alignment. Primers were then designed according to this sequence information. PCRs were performed for genomic DNA isolation of the porcine Myd88 gene. PCR profiles were 5 min at 95 °C, followed by 35 cycles of 30 s at 94 °C, 30 s at annealing temperature, 90 s at 72 °C and a final extension of 5 min at 72 °C. All PCR products were sequenced by commercial services.

Chromosome mapping of the porcine Myd88 gene

The radiation hybrid (RH) panel was used for porcine Myd88 gene chromosome mapping analysis (Yerle et al., 1998). PCR was performed in a 10 mL reaction mixture containing 25 ng of cell hybrid line DNA, a 1x PCR buffer (TaKaRa, Dalian, P. R. C), 0.3 mM of each primer (Table 1), 75 mM of each dNTPs, 1.5 mM MgCl2 and 1 U Taq DNA polymerase (TaKaRa, Dalian, P. R. C). The PCR profile was 5 min at 94 °C followed by 35 cycles of 30 s at 94 °C, 30 s at 61 °C, 40 s at 72 °C, and a final extension of 5 min at 72 °C. PCR results were analyzed using the IMpRH mapping tool (Milan et al., 2000).

 

 

Tissue distribution of the porcine Myd88 gene

For tissue distribution analysis, eleven tissues including heart, liver, spleen, lung, kidney, fat, skeletal muscle, lymph node, small intestine, large intestine and brain were obtained from four 18 to 24-months-old Wuzhishan mini-pigs. Total RNA was extracted from each sample using the TRIzol reagent (Invitrogen, San Diego, CA), and then treated with RNase-free DNase I (MBI Fermentas, Germany). RNA concentration was determined, and equal amounts of RNA from each targeted tissue sample from different individuals were mixed to form the RNA pool. The RNA pool from each sample was reverse-transcribed into cDNA by means of M-MLV reverse transcriptase (Promega, USA). Real-time PCR was performed in a 20 mL mixture containing 1x PCR buffer (TaKaRa, Dalian, P. R. C), 3.0 mM MgCl2, 100 µM each dNTP, 0.3 µM gene-specific primers (Table 1), 0.3x SYBR Green I, 2 U Taq DNA polymerase (TaKaRa, Dalian, P. R. C), and 2 µL template cDNA. Reactions were carried out in an Opticon 2 real-time cycler (MJ Research, Waltham, MA), the cycling conditions consisting of an initial 5 min at 95 °C, followed by 35 cycles of 15 s at 95 °C (for denaturation), 30 s at 65 °C (for annealing), 30 s at 72 °C (for elongation) and fluorescence acquisition at 83 °C for 1 s. PCR was performed in triplicate and gene expression levels were quantified relative to the expression of endogenous β-actin, by using Gene Expression Macro software (Bio-Rad, Richmond, CA) employing an optimized comparative Ct (DDCt) value method. Expression was considered undetectable when the Ct value of the targeted gene exceeded 35.

SNPs identification and association analysis

DNA samples from seven breeds, including three Chinese indigenous (Wuzhishan, Laiwu, Bamaxiang and Guizhouxiang) and two foreign breeds (Landrace and Yorkshire), were used as PCR templates for Myd88 genomic DNA isolation. All PCR products were sequenced. Subsequently, all sequenced information related to the porcine Myd88 gene, this including our PCR results, the ESTs and genomic DNA fragments available on NCBI, was used to analyse potential SNPs. A potential SNP site was considered as that where different alleles appeared more than twice.

Genetic variation was studied in seven unrelated breeds of pigs, namely, Tongcheng, Wuzhishan, Laiwu, Bamaxing, Guizhouxiang, Yokshire and Landrace. The experimental group underwent association analysis. This group consisted of three pure-blood populations, Tongcheng (T), Landrace (L) and Yorkshire (Y), and two crossbred populations, LYT (L male x YT female) and YLT (Y male x LT female). Six porcine immune-traits were examined. These were red blood cell count (RBC), hematocrit (HCT), mean corpuscular volume (MCV), IgG, blood cell distribution width (RDW) and delayed-type hypersensitivity (DTH). In order to determine immune-traits, the blood from 20-weeks-old pigs was collected so as to detect RBC, HCT, MCV and RDW, by using a blood cell auto-analyzer (MEK-5216K). IgG concentration was ascertained through the radial immuno-diffusion method. The Delayed-type hypersensitivity (DTH) trait was detected by means of the phytohemagglutinin (PHA) skin test, according to the van Heugten method, with a minor modification (Van Heugten et al., 1994).

A general linear model (GLM) was used to estimate the association between genotypes and immune traits. According to the structure of the population, the model used for trait association analysis is described as follows:

Yijk = µ + Pi + Gj + Bk + (PG)ij + (PB)ik + (GB)jk + eijk

where Yijkl = lth trait measured in the animal; µ = overall mean; Pi = fixed effect of the ith population (i = 1, 2, 3, 4, 5); Gj = fixed effect of the jth genotype (j = 1, 2, 3); Bk = fixed effect of the kth batch (phenotypic data were recorded in two periods, k = 1, 2); (PG)ij = effect of interaction ith population jth genotype; (PB)ik = effect of interaction ith population kth batch; (GB)jk = effect of interaction jth genotype kth batch; eijkl = error term.

Phylogenetic tree and structure analysis

Myd88 proteins from many species were collected for phylogenetic tree analysis and the homologues of the sequences analyzed by means of the ClustalW program. A phylogenetic tree was retrieved by using MEGA 3.1 software. Conserved residues of the functional domain of porcine Myd88 were analyzed through multiple sequences alignment. The three-dimensional (3-D) model was predicted through the 3djigsaw program. An image of the 3-D model was obtained by using software Raswin 2.7 software. Phosphorylation sites were predicted by the NetPhos program.

 

Results and Discussion

Isolation and chromosome assignment

The DNA segment isolated was 4464 bp, subsequently deposited in to GenBank (GenBank no, EU056737). The isolated genomic sequence contained the complete ORF (882 bp) of the porcine Myd88 gene (NM_001099923). RH mapping results revealed that this gene was assigned to the long arm of the pig chromosome 13 (SS13q), the closest linked marker being S0288 (distance = 40 cR; LOD = 8.66). In humans, Myd88 has been mapped on 3p22 (Bonnert et al., 1997). Comparative genomic analysis results confirmed that pig chromosome 13 is homologous with human chromosome 3 (Sun et al., 1999). Thus, our mapping results conformed to those from comparative genomic analysis.

Detection of tissue distribution

Real time-PCR analysis was performed to determine the mRNA expression profile of the Myd88 gene in Wuzhishan mini-pigs. The data revealed Myd88 gene expression in all examined tissues, this expression being relatively low in skeletal muscle tissue (Figure 1). Previous studies also showed wide Myd88 gene expression in digestive tissues, the spleen and mesenteric lymph nodes (Tohno et al., 2007). In humans, Myd88 was found to be constitutively expressed in many tissues (Hardiman et al., 1996), this thus implying the similarity of the tissue distribution profile of the porcine Myd88 gene to that in humans. TLRs are widely expressed in many tissues (Zarember and Godowski, 2002), and Myd88 functions as the adaptor protein of TLRs. Moreover, sub-cellular localization results confirmed that Myd88 found in cytoplasm was not a secretory protein (Nishiya et al., 2007). Therefore, Myd88 needs to be widely expressed in order to participate in TLR signal transduction.

 

 

Polymorphism detection and association analysis

Porcine Myd88 gene polymorphisms were detected by multiple sequence comparison. According to our results, the 4464 bp genomic DNA of Myd88 contained 16 potential SNPs which were 797T/C, 813A/g, 1721T/g, 1755C/A, 2130T/A, 2461C/T, 2468G/A, 2519G/A, 2743A/g, 2757C/T, 3076A/g, 3258A/g, 3291T/A, 3298C/T, 3345A/g and 3485G/A. None of these resulted in residual changes, this indicating that the porcine Myd88 protein was highly conserved. 797T/C polymorphism of the porcine Myd88 gene, which can be detected by the PCR-RFLP method, was further studied. The genotypes of this site were identified by using the restriction enzyme ApaL I (TT 185 bp, CC 160/25 bp, TC 185/160/25 bp) (Figure 2). Allele frequency analysis revealed a much higher frequency of allele T in five Chinese indigenous breeds than in Landrace and Yorkshire (Table 2). We performed a preliminary association study to determine whether this polymorphism had affected any immune-traits in the pig. The data showed that there was no significant association between this SNP and immune traits RBC, HCT, MCV, IgG, DTH and RDW (p > 0.05) (Table 3).

 

 

 

 

 

 

Phylogenetic tree and structural characterization

The porcine Myd88 protein contained 293 residues with an overall sequence similarity to Myd88 in human (88%), chicken (70%), clawed frog (65%), zebrafish (62%) and sea urchin (40%). Phylogenetic tree analysis also showed that Myd88 was conserved during evolution (Figure 3). Highly conserved residues of Myd88 were detected through sequence comparison with the five species mentioned above. Porcine Myd88 contained two functional domains, DD (residues: 19-109) and TIR (residues: 157-293) (Tohno et al., 2007), the most abundant amino acid in DD being Leu (19.8%). 17 DD residues were conserved in all the species examined. Among the conserved residues, there were 7 Leu residues and 12 hydrophobic residues (A, I, L, F, W, and V belong to hydrophobic amino acid). These results indicate that hydrophobic residues, especially of Leu, may play important roles in maintaining DD structure and functioning.. The 3-D model of the porcine Myd88 death domain was predicted using the 3djigsaw program. According to the model, the hydrocarbon chains of Leu residues were packed in the inner part of the DD, thus forming a hydrophobic interior. The highly conserved Leu residues (Leu33, 75, 89, 90, 105) formed a Leu plane (Figure 4). These results also indicate that Leu residues may play important roles in the death domain.

 

 

 

 

The other functional domain of porcine Myd88 is a TIR containing 137 residues. A BB-loop, which was found in the TIR domain of TLRs (Xu et al., 2000), was also found in the TIR of the porcine Myd88 in our studies. This loop contained the motif (RDxLPG, x represents L or V), and was found to be highly conserved among all the species studied. Previous research has confirmed that the BB-loop was essential for maintaining theTLR4 function. Substitution of the Pro residue in the BB-loop of TLR4 by His abolished the TLR4 immune-response to lipopolysaccharide (Poltorak et al., 1998). The BB-loop, highly conserved during evolution, may be very important for the porcine Myd88 signaling pathway. In addition, another conserved motif (CDFQTKFAxSL, x represents L or V) was found in the TIR of porcine Myd88 (Box 2). This motif contains a conserved Ser which may be a phosphorylation site predicted through using the NetPhos program (p = 0.959), and may be related to phosphorylation of porcine Myd88. A 3-D model of the TIR of porcine Myd88 was predicted by using the 3djigsaw program (Figure 5), the conserved domains being labeled in yellow and the Ser in Box 2 in blue.

 

 

Acknowledgments

We are grateful to Dr. Yerle for supplying the RH panel. This research was supported by the State Platform of Technology Infrastructure (2005DKA21101), the Key Project of National Basic Research and Developmental Plan of China (G2006CB102105), the National High Science and Technology Foundation of China (20060110Z1039), the National Natural Science Foundation of China (30571300), the National Scientific and Technology Mainstay Project (2006BDA13B08) and the Project of Science and Technology Innovation Team for "Research and Improvement of Domestic Animal Germplasm" of IAS, CAAS.

 

References

Adachi O, Kawai T, Takeda K, Matsumoto M, Tsutsui H, Sakagami M, Nakanishi K and Akira S (1998) Targeted disruption of the MyD88 gene results in loss of IL-1- and IL-18-mediated function. Immunity 9:143-150.         [ Links ]

Bonnert TP, Garka KE, Parnet P, Sonoda G, Testa JR and Sims JE (1997) The cloning and characterization of human MyD88: A member of an IL-1 receptor related family. FEBS Lett 402:81-84.         [ Links ]

Hardiman G, Rock FL, Balasubramanian S, Kastelein RA and Bazan JF (1996) Molecular characterization and modular analysis of human MyD88. Oncogene 13:2467-2475.         [ Links ]

Lemaitre B, Nicolas E, Michaut L, Reichhart JM and Hoffmann JA (1996) The dorsoventral regulatory gene cassette spatzle/Toll/cactus controls the potent antifungal response in Drosophila adults. Cell 86:973-983.         [ Links ]

Li C, Zienkiewicz J and Hawiger J (2005) Interactive sites in the MyD88 Toll/interleukin (IL) 1 receptor domain responsible for coupling to the IL1beta signaling pathway. J Biol Chem 280:26152-26159.         [ Links ]

Li L, Cousart S, Hu J and McCall CE (2000) Characterization of interleukin-1 receptor-associated kinase in normal and endotoxin-tolerant cells. J Biol Chem 275:23340-23345.         [ Links ]

Medvedev AE, Lentschat A, Wahl LM, Golenbock DT and Vogel SN (2002) Dysregulation of LPS-induced Toll-like receptor 4-MyD88 complex formation and IL-1 receptor-associated kinase 1 activation in endotoxin-tolerant cells. J Immunol 169:5209-5216.         [ Links ]

Medzhitov R and Janeway Jr CA (1997) Innate immunity: The virtues of a nonclonal system of recognition. Cell 91:295-298.         [ Links ]

Milan D, Hawken R, Cabau C, Leroux S, Genet C, Lahbib Y, Tosser G, Robic A, Hatey F, Alexander L, et al. (2000) IMpRH server: An RH mapping server available on the Web. Bioinformatics 16:558-559.         [ Links ]

Nishiya T, Kajita E, Horinouchi T, Nishimoto A and Miwa S (2007) Distinct roles of TIR and non-TIR regions in the subcellular localization and signaling properties of MyD88. FEBS Lett 581:3223-3229.         [ Links ]

Palladino MA, Johnson TA, Gupta R, Chapman JL and Ojha P (2007) Members of the toll-like receptor family of innate immunity pattern-recognition receptors are abundant in the male rat reproductive tract. Biol Reprod 6:958-964.         [ Links ]

Poltorak A, He X, Smirnova I, Liu MY, Van Huffel C, Du X, Birdwell D, Alejos E, Silva M, Galanos C, et al. (1998) Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: Mutations in Tlr4 gene. Science 282:2085-2088.         [ Links ]

Scanga CA, Aliberti J, Jankovic D, Tilloy F, Bennouna S, Denkers EY, Medzhitov R and Sher A (2002) Cutting edge: MyD88 is required for resistance to Toxoplasma gondii infection and regulates parasite-induced IL-12 production by dendritic cells. J Immunol 168:5997-6001.         [ Links ]

Sun HF, Ernst CW, Yerle M, Pinton P, Rothschild MF, Chardon P, Rogel-Gaillard C and Tuggle CK (1999) Human chromosome 3 and pig chromosome 13 show complete synteny conservation but extensive gene-order differences. Cytogenet Cell Genet 85:273-278.         [ Links ]

Takeda K and Akira S (2004) TLR signaling pathways. Sem Immunol 16:3-9.         [ Links ]

Tohno M, Shimazu T, Aso H, Kawai Y, Saito T and Kitazawa H (2007) Molecular cloning and functional characterization of porcine MyD88 essential for TLR signaling. Cell Mol Immunol 4:369-376.         [ Links ]

Uematsu S and Akira S(2006) Toll-like receptors and innate immunity. J Mol Med 84:712-725.         [ Links ]

Van Heugten E, Spears JW, Coffey MT, Kegley EB and Qureshi MA (1994) The effect of methionine and aflatoxin on immune function in weanling pigs. J Anim Sci 72:658-664.         [ Links ]

Xu Y, Tao X, Shen B, Horng T, Medzhitov R, Manley JL and Tong L (2000) Structural basis for signal transduction by the Toll/interleukin-1 receptor domains. Nature 408:111-115.         [ Links ]

Yamamoto M and Akira S (2004) TIR domain-containing adaptors regulate TLR-mediated signaling pathways. Nippon Rinsho 62:2197-2203.         [ Links ]

Yerle M, Pinton P, Robic A, Alfonso A, Palvadeau Y, Delcros C, Hawken R, Alexander L, Beattie C, Schook L, et al. (1998) Construction of a whole-genome radiation hybrid panel for high-resolution gene mapping in pigs. Cytogenet Cell Genet 82:182-188.         [ Links ]

Zarember KA and Godowski PJ (2002) Tissue expression of human Toll-like receptors and differential regulation of Toll-like receptor mRNAs in leukocytes in response to microbes, their products, and cytokines. J Immunol 168:554-561.         [ Links ]

 

Internet Resources

The IMpRH mapping tool, http://IMpRH.toulouse.inra.fr/.         [ Links ]

The clustal w program, http://www.ebi.ac.uk/clustalw/.         [ Links ]

The 3djigsaw program, http://www.bmm.icnet.uk/servers/3djigsaw/.         [ Links ]

The NetPhos program, http://www.cbs.dtu.dk/services/NetPhos/.         [ Links ]

 

 

Send correspondence to:
Kui Li
Institute of Animal Science, Chinese Academy of Agricultural Sciences
100094 Beijing, P.R. China
E-mail: kuili@iascaas.net.cn

Received: June 6, 2008; Accepted: November 24, 2008.

 

 

Associate Editor: Luiz Lehmann Coutinho

Creative Commons License All the contents of this journal, except where otherwise noted, is licensed under a Creative Commons Attribution License